Equine encephalomyelitis vaccine



Patented June 11, 1940 2,204,064

UNITED STATES PATENT orrice EQUINE ENCEPHALOMYELITIS VACCINE Joseph W. Beard, Durham, N. O.

No Drawing. Application May 13, 1938. Serial No. 207,685

6 Claims. (Cl. 167-78) This invention relates to equine encephalomyener in order to make thorough extraction of the litis vaccine (either eastern or western strain) embryo tissues possible, the mixture of solid and and to methods of preparing the same. liquid obtained by grinding the embryo being In the past, vaccines for practical use in the referred to as ground embryo sap. Thus the immunization of horses against equine encephembryos may be ground up with sand in any 5 alomyelitis have been prepared from diseased suitable device. The ground embryo sap is then horse brains containing the virus. These vacdiluted or suspended in 0.9 per cent sodium cines, however, leave much to be desired because chloride solution to a 20 per cent suspension based of the low content and variable concentration on the original weight of the embryos. To the 10 of the virus as produced in horse brains. 20 per cent suspension of embryo sap an equal 10 The virus of equine encephalomyelitis has been volume of 0.8 per cent formalin solution prepared grown also in chick embryos. in 0.9 per cent sodium chloride solution is added I have discovered that I may obtain a highly yielding a final concentration of 0.4 per cent effective vaccine in substantial quantities from formalin in a 10 per cent suspension of chick l5 equine encephalomyelitis virus that has been embryo tissue. The suspension is well mixed and propagated in the living tissues of fowl embryo. is stored in a refrigerator until the mixture no By my process the virus so produced is inactivated longer contains infectious virus detectable by any to yield a vaccine on a scale sufficiently large for of the known methods. This is accomplished and commercial distribution for the vaccination of the suspension i ady o se as 1 vaccine in horses, for instance, against natural infection five to seven days. 20 from the disease. 7 However, the suspension of vaccine may be The following specific example given by way p fi d in any n of a n m f ways t y ld of illustration and not in limitation describes one a e Potent Product and One more easily specific method of preparing my vaccine. handled. The active immunizing material can Fertile chicken eggs are incubated in an incue p p e a pa y p fi d y the usual 25 bator or warm room at such a temperature and chemical methods utilized in the precipitation, under such conditions that the embryo will dec n ntra n and p rific n of pr t in matevelop normally. This temperature is usually rial. Another manner in which the immunizing about 103 F. When the eggs have been in umaterial may be concentrated and purified with so bated for ten days, an opening is made in the l le l s in potency is by means f ultrashell with a grinder or by some other suitable centrifuge. Any th r su t e cal and/0r means. A small amount of the virus-containing p ys c e ods ay be u d or Concentramaterial or inoculum is introduced through the tion and Purification the V cc n opening onto the chorio-allantoic membrane of In tests With y Vaccine for the mm ni ation the embryo. The shell is then sealed with of various animals, I have found that the mate- 35 cover glass or other means and the egg is rerials p p d in accordance With my p c ss turned to the incubator. Obviously the inoculaave sh w a high degree 0f c iv ness. With tion is carried out in such a manner as not to uin a p of e u fl gh 1- cc. of interfere with the development of the chick. v ccine prep re as r bed ab ve w injected 40 The eggs containing the inoculated embryos subcutaneously followed by a similar injection 0 are left in the incubator until just before they Seven y after the fi These jec /1 115 imdie, usually about thirteen hours after inoculamunized the guinea D against n r ous doses tion, I fe tion of th embryo by in l ti i of infectious virus injected into the brain fourteen practically always followed by the death of the ys or o g a r the last injection o y VaC- 5 embryo if it is permitted to remain in the incue. h s b done repeatedly nd n no bator for a sufilcient period of time. By watchinstance has a gu a p receiving y acc ne ing the embryo, however, it is possible to predict been lost even when the infecting dose has been to within a few minutes of when it will die as 1000 tim s he minimum fatal dose- In testing indicated by the slowing movement of the my vaccine on horses, I have also obtained defiembryo. nite immunity against many minimum fatal doses 50 In order to obtain the maximum yield of virus of virus injected intracerebrally. and vaccine, the embryos are removed from the My method of preparation of the vaccine is shells as near as possible to the time just before suitable for the eastern strain virus as well as they are to die. The removed embryos are imthe western strain virus. Apparently the vaccine mediately chilled, and then ground insome manprepared from embryos infected with eastern 55 strain virus protects only against infection with eastern strain virus and vaccine from embryos diseased with western strain virus protects only against infection with western strain virus.

In place of the chick embryos specifically described I may utilize in my method the embryo of other fowl such as embryos of ducks, geese, turkeys, guinea-fowls, etc. However, chicken eggs are probably the cheapest and the most easily obtainable.

The exact age of the embryos is not of major importance, I have used nine to fourteen day old embryos with success. The embryo may be inoculated with the virus in any suitable way other than that specifically described in the example. Thus the virus may be introduced into the embryo by intravenous injection, injection directly into the embryo itself or by some other suitable method. The time for incubation of the inoculated embryo may be varieci somewhat although, for maximum yield, I have found it best to remove the embryo from the shell as near as possible to the time just before it dies.

In place of the sodium chloride solution used as a diluent, I may use other materials such as plain water or other types of diluting solutions. The concentration to which the ground embryo sap is diluted may be varied and likewise the concentration of formalin is not especially criticai, at least not in principle. From a practical point of View and in order to obtain the maximum yield, the amount of formalin which is used should be suificient to inactivate all of the active infectious virus but should not be sufficiently concentrated to destroy the immunizing capacity of the vaccine against the active infectious virus. In place of formalin inactivation I may utilize other chemical or physical methods such as light, heat, etc. The time of inactivation is not necessarily fixed but may easily be determined under any given circumstances.

The active infectious virus of either the eastern or western strain is obtained from any suitable source such as horse brain, guinea pig or mouse brain, chick embryo tissues or any other tissues or materials containing infectious virus. This virus is customarily utilized as a suspension as a convenient form for infection of the embryo.

i, To produce a suitable suspension the tissues containing the active virus may be ground with sand and diluted or made up with sodium chloride solution, hormone broth, or the like. The exact medium is not important so long as the inocuilum is sterile with respect to bacteria and contains sufficient active virus to infect the embryo.

The above description has referred to a vaccine grown on the living developing whole chick embryo. The procedure would doubtless be equally applicable to virus cultures prepared from other forms of living embryo tissues.

Any other suitable changes or variations may be made in carrying out the process of my invention without departing from the spirit and scope except as defined in the appended claims.

I claim:

1. An equine encephalomyelitis vaccine cornposition comprising formalinized fowl embryo tissues which had been diseased with equine encephalomyelitis, said composition being capable of immunizing horses against equine encephalomyelitis without substantially inducing the disease.

2. An equine encephalomyelitis vaccine composition comprising inactivated fowl embryo tissue which had been diseased with equine encephalomyelitis, said composition being capable of immunizing horses against equine encephalomyelitis without substantially inducing the disease.

3. An equine encephalomyelitis vaccine composition comprising a formalinized suspension of fowl embryo tissue which had been diseased with equine encephalomyelitis, said composition being capable of immunizing horses against equine encephalomyelitis without substantially inducing the disease.

4. A process for the production of equine encephalomyelitis vaccine which comprises infecting living fowl embryo tissue with the infectious virus of equine encephalomyelitis, propagating the virus in the tissue, and inactivating the virus so propagated without substantially destroying the immunizing capacity of the resulting vaccine.

5. A process for the production of equine encephalomyelitis vaccine'which comprises infecting a living fowl embryo with the infectious virus of equine encephalomyelitis, incubating the embryo until just prior to the death thereof whereby the virus is propagated, removing the embryo, chilling the embryo, extracting the embryo sap, and inactivating the infectious virus in the sap without substantial destruction of the immunizing capacity of the vaccine produced by the inactivation.

6. A process for the production of equine encephalomyelitis vaccine which comprises infect ing a living fowl embryo with the infectious virus of equine encephalomyelitis, incubating the embryo until just prior to the death thereof whereby the virus is propagated, removing the embryo, chilling the embryo, grinding the embryo, suspending the ground embryo in a weak salt solution, and inactivating the virus by adding formaldehyde in amount sufficient for inactivation but insuficient to cause any substantial destruction of the immunizing capacity of the vaccine so produced.

JOSEPH W. BEARD. 

